Cold Spring Harbor Protocols

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ISSN / EISSN : 1940-3402 / 1559-6095
Published by: Cold Spring Harbor Laboratory (10.1101)
Total articles ≅ 9,796
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Michael R. Green, Joseph Sambrook
Cold Spring Harbor Protocols, Volume 2021;

In this method, a short primer is hybridized to an oligonucleotide template whose sequence is the complement of the desired radiolabeled probe. The primer is then extended using the Klenow fragment to incorporate [α-32P]dNTPs in a template-directed manner. After the reaction, the template and product are separated by denaturation followed by electrophoresis through a polyacrylamide gel under denaturing conditions. With this method, it is possible to generate oligonucleotide probes that contain several radioactive atoms per molecule of oligonucleotide and to achieve specific activities as high as 2 × 1010 cpm/µg of probe. Because the end product of the reaction is dsDNA, whose strands must be separated and the labeled product isolated, this method is generally not used to prepare nonradiolabeled oligonucleotides.
Edward A. Greenfield
Cold Spring Harbor Protocols, Volume 2021;

Smaller animals such as rats, mice, hamsters, and guinea pigs are usually poor choices for polyclonal antibody production because only small volumes of serum can be obtained. This problem can be reduced by inducing the formation of ascites in mice, which can provide up to 10 mL of ascites fluid from a single animal. Antibody titers in ascites fluids are almost as high as serum titers.
Michael R. Green, Joseph Sambrook
Cold Spring Harbor Protocols, Volume 2021;

When labeled oligonucleotides are to be used in enzymatic reactions such as primer extension, virtually all of the unincorporated label must be removed from the oligonucleotide. For this purpose, chromatographic methods or gel electrophoresis are superior to differential precipitation of the oligonucleotide with ethanol or cetylpyridinium bromide (CPB). This protocol describes a method to separate labeled oligonucleotides from unincorporated label that takes advantage of differences in mobility between oligonucleotides and mononucleotides during size-exclusion chromatography. Although size-exclusion chromatography can, in principle, be used to purify either radiolabeled or nonradiolabeled oligonucleotides, this protocol is geared toward purifying radiolabeled oligonucleotides, whose elution from the column is monitored using a minimonitor and whose separation from unincorporated nucleotides is monitored by liquid scintillation counting.
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